At least 2 cDNAs of the sialyltransferase (ST) gene family have been cloned and the promoters tor two glycoprotein ST genes partially characterized. However, no information is available regarding the promoter sequence for glycolipid ST genes. We now report the characterization ot the promoter sequence for rat CMP-NeuAc:GM3 "2,8 sialyltransfcrase (GD3-synthase) gene. Based on the cDNA sequence for rat GD3symhasc (BBRC. 226:319. 1996), the 5'-untranscribed DNA fragments of the cDNA were oh t dined by the genomic walking method. Five pools of the uncfoned rat genomic DNA were used as the (empiètes for PCR. The sense primers were derived from the adaptor sequence ol the genomic DNA pools and the antisense gene-specific primers trom CiD.Vsynthase cDNA. After two rounds of PCR amplification, two major bands of 0 4 and 1 1 kb were obtained. DNA sequencing indicated that the 0.9 kb fragment was identical to the V end of the 2.2 kb fragment and both were immediately upstream of the (il)3-t;ynthasc cDNA. DNA sequencing also revealed the presence of potential binding sues tor Sp-1, AP-2, ATF. and NF-1. These fragments were then subcloned into pfigalBasic and pttgal-Enhanccr vector m both the forward and reverse orientations. The expression level of β-galactosidase was used to assess the promoter activity after Iranstection of the constructs to F-11 cells, a hybrid cell line of rat dorsal root ganglion cells \mouse neuroblastoma N18TG-2 cells, which expresses a high level of GD3. The en/ynie activity of the constructs containing the 0.9- and 2.2-kb fragments in the forward orientation was 8- to 11-fold, respectively, over that of the pβgal-Basic vector alone in 1-11 cells These data suggest that the promoter activity is in fact located in the cloned 1'iagmenis directly upstream of the first exon in the GD3-synthase gene. The constructs containing (he inserts in reverse orientation had little of the β-galactosidasc activity in the cell1-, indicating that the cloned fragments contained the promotor for a single gene.
|Original language||English (US)|
|Publication status||Published - Dec 1 1997|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology